Ubiquitination is a common pathway for multiple cellular signals including protein turnover via degradation in the proteasome. Defects in the signaling system have been directly implicated in a range of diseases including neurodegenerative disorders such as Parkinson's, and a range of human cancers including breast and ovarian. The ubiquitination process involves a cascade of enzymes to activate and transfer ubiquitin (Ub) to a substrate. In the final step, the E3 ubiquitin ligase acts to localize both a substrate and ubiquitin-conjugated E2. Recent data support the theory that in most cases a specific E2/E3 pair will either monoubiquitinate a substrate, or polyubiquitinate a previously ubiquitinated substrate. CHIP (carboxylterminus of Hsc70 interacting protein) is a U-box containing E3 ubiquitin ligase that interacts with chaperone proteins and is involved in the cellular response to stress. A yeast-two hybrid screen for investigating E2 binding partners for a specific E3 has been established, and will be used to investigate interactions with CHIP (Aim 1). NMR will then be used to confirm and map the interactions, and function will be tested by in vitro ubiquitination assay. Aim 2 focuses on the mechanism whereby the ubiquitin-bound E2 is activated for ubiquitin transfer by interaction with an E3 ligase. Functional assays support a mechanism in which binding to an E3 is required for E2~Ub to function in ubiquitination transfer;however there is no structural information available for any E2~Ub/E3 complex. An active site mutant E2 will be used to stabilize the Ubcharged form, and three separate series of titrations will be performed in analysis of the ternary complex: one protein will be isotopically labeled in each experiment. Understanding the function of E2/E3 complexes will greatly facilitate attempts to assign E2 and E3 proteins involved in specific signaling pathways. These studies will assist to define the interactions of CHIP with E2 machinery both in E2 specificity and the mechanism involved in stimulation of ubiquitin transfer. PUBLIC HEALTH RLEVANCE: Ubiquitination is a common cellular signaling pathway which has been directly implicated in a range of diseases. This work to define specific functional interactions between components of the ubiquitination machinery is a necessary step in monitoring and treating system failures which lead to disease.